Gut Reaction: New Locale for Antibody Activity

Most antibodies do their work in the bloodstream. But others maybe powerless to knock out their disease-causing foes unless theconfrontation takes place inside an intestinal cell, researchershave found.

The unexpected discovery suggests a novel approach for designingantibody-stimulating vaccines against viruses that infect throughthe gut, said Dr. Harry Greenberg, a medical investigator at theVeterans Affairs Palo Alto Health Care System and chief of thedivision of gastroenterology at Stanford University School ofMedicine.

The finding also explains the surprisingly broad effectivenessof a rotavirus vaccine invented by Greenberg and colleagues whenhe worked at the National Institute of Allergy and InfectiousDiseases.

Greenberg and three Stanford colleagues describe the researchin the April 5, 1996, issue of Science. A "Perspectives"article explaining the significance of this work appears in thesame issue.

The Stanford researchers made the finding during a study of immunityto rotavirus, the major cause of childhood diarrhea. Greenbergis associate chair of Stanford's Department of Medicine and aprofessor of gastroenterology.

A Costly Malady

Diarrhea due to rotavirus kills between 800,000 and 1 millionchildren annually, mainly in Third World countries. In the UnitedStates, this virus causes few deaths but costs over $1 billioneach year as a result of health-care expenses and days missedfrom work, Greenberg said.

"When you have something as damaging as this, you have abasic reason to want to understand immunity to the virus. Youhope that understanding immunity will better equip you to producea successful vaccine," Greenberg said.

Many previous studies of rotavirus immunity were carried out invitro in artificial settings such as petri plates, Greenberg said.His group set out to learn how rotavirus immunity works in livingorganisms.

In one experiment, Greenberg and colleagues fed infectious rotavirusto mice they had treated to produce large quantities of antibodiesto rotavirus proteins. They attached an antibody-producing tumorto each mouse's back to mimic the natural production of immunoglobulinA (IgA) antibodies.

Unlike IgG antibodies--the type usually stimulated by vaccines--IgAcan attach to the special epithelial cells lining the intestines,enter them, and then pass into the gut. These epithelial cellsmake up a mucous membrane.

In the experiment, each tumor was designed to produce antibodiesagainst a single rotavirus protein.

The researchers gauged how well the antibodies worked by measuringthe amount of virus in the animals' feces: A little virus indicatedthat the antibody killed the virus before it could reproduce toa great extent; a lot meant that the antibody failed to stop thevirus.

The researchers were surprised to find that antibodies to oneprotein, called VP6, were the only ones that thwarted the virus.This was unexpected because in the earlier in vitro studies, antibodiesto VP6 had never been successful.

Also surprising was the finding that the antibody to protein VP4,which had stopped the virus in the in vitro studies, failed toknock out the infection in the mice.

"What we're saying is, in this model, unexpectedly, antibodiesthat had no activity in vitro were highly protective in vivo,"Greenberg said.

Next, the researchers tried placing the VP6 antibodies directlyin the gut. This time the antibodies had no effect.

What seems to be going on here, said Greenberg, is that some antibodies,such as those against VP6, work only when they meet their targetinside an intestinal cell. Something happens after they enterthe cell that affects their ability to neutralize the virus.

Implications for Vaccine Design

Since most vaccines stimulate only IgA antibodies, which are unableto pass through epithelial cells, this finding might prompt vaccinedesigners to rethink their strategy for fighting the many pathogens--rangingfrom the virus that causes the common cold to HIV--that infectthrough mucous membranes, Greenberg said.

"If what we've shown is a generally applicable phenomenon,it means the host immune system might have another way of interruptingthe many pathogens that infect at mucous membranes--a way thatvaccine designers have not really looked at," Greenberg said.

The effect of location on the activity of certain antibodies mightexplain the surprisingly broad effectiveness of the rotavirusvaccine invented by Greenberg and colleagues. Although the experimentalvaccine was expected to protect against one type of rotavirus,it appears to protect against several, Greenberg said. The vaccineis likely to be under study by the FDA in the near future foruse in the United States, he said.

What had mystified researchers about the vaccine was that it couldstop viral variants it was not designed to thwart.

"We find that rotavirus comes in different serotypes, or'flavors.' Some flavors have one type of a protein called VP7,others have a different type," Greenberg said. The vaccineworked against viruses carrying various types of VP7, even thoughit stimulated antibodies against only one type of VP7.

Now Greenberg suspects that VP6 holds the answer to the paradox.

Although the experimental vaccine stimulates antibodies againstVP6 and VP7, researchers had assumed that VP6 antibodies playedno role in stopping infection because VP6 had been ineffectivein the in vitro studies.

But Greenberg's new study shows that VP6 antibodies can stop infectionin vivo. And unlike VP7, VP6 invades very little from one virusto the other.

"It may well be the antibodies against VP6 stimulated bythe vaccine that are granting immunity," he said.

Greenberg's coauthors were Stanford postdoctoral fellows JohnW. Bums and Majid Siadat-Pajouh and undergraduate Ajit A. Krishnaney.

The research was funded, in part, by the National Institutes ofHealth, the World Health Organization, and the Department of VeteransAffairs.